Project Summary HIV infection begins with fusion of viral and target cell membranes, driven by the viral envelope glycoprotein [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3]. Gp120 binding to primary receptor CD4 and coreceptor (e.g. chemokine receptor CCR5 or CXCR4) trigger large structural rearrangements in gp41 that drive the membrane fusion process. Encounter of the coreceptor by gp120 is believed to be the crucial trigger for gp41 refolding events, which promotes membrane fusion. It has been two decades since CCR5 and CXCR4 were first identified as the coreceptors for HIV-1 entry, but we still do not have a clear picture, in particular, at atomic resolution, of how the coreceptor recognizes HIV-1 Env, except for some speculative molecular modeling. The coreceptors are chemokine receptors with seven transmembrane-spanning segments (7TMs) and belong to the family of G protein-coupled receptors (GPCRs). Crystal structures have been reported for heavily modified CCR5 and CXCR4, revealing the general architecture of these receptors, as well as their interactions with the various ligands, but the structures fall short of explaining the molecular details of how these chemokine receptors function as HIV-1 coreceptors. In this proposal, we plan to gain a better understanding of the HIV-1 coreceptor function of CCR5 and CXCR4 and to provide high-resolution pictures of how they interact with HIV- 1 Env to promote viral entry. We hypothesize that an extensive interface between gp120 and the coreceptor involving multiple structural elements is required for their high-affinity interaction. We have already purified a stable complex of HIV-1 gp120, 4 domain CD4 and an unmodified human CCR5, and demonstrated the feasibility to carry out electron microscopy (EM) studies. Recent advances in cryo-electron microscopy (cryoEM) have revolutionized the field of structural biology and produced numerous high-resolution structures. To capitalize on these advances, we will tackle a challenging problem that is important to both the HIV and GPCR fields. We will pursue following specific aims: 1) we will determine the high-resolution structure of the complex of CD4-gp120-CCR5 by cryoEM; 2) we will determine the high-resolution structure of the complex of CD4-gp120-CXCR4; 3) we will elucidate the role of key structural elements of CCR5 or CXCR4 in coreceptor function by structure-guided mutagenesis.